Multi-view imaging apparatus

ABSTRACT

Systems and methods are provided for taking images of a sample. The sample is placed in an imaging box comprising a moveable stage that allows images of the sample to be taken from various positions and angles within the imaging box. The images are taken by a camera and sent to a processor. Structured light images obtained from one or more views within the imaging box may be used to build a structured light representations of the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under U.S.C. §120 from co-pending U.S.patent application Ser. No. 09/905,668, filed Jul. 13, 2001 andentitled, “MULTI-VIEW IMAGING APPARATUS”, which is incorporated hereinfor all purposes.

FIELD OF THE INVENTION

The present invention relates generally to imaging systems and theirmethods of use. More specifically, the present invention relates toimaging systems and methods used in capturing images from multipleviews.

BACKGROUND OF THE INVENTION

One specialized type of imaging involves the capture of low intensitylight (on the order of individual photons) from a light emitting sample,and the construction of images based on the photon emission data. Thissource of light in the sample visually indicates the origin of theactivity of interest. For example, specialized in-vivo imagingapplications may include analysis of one or more representations ofphoton emissions from internal portions of a specimen superimposed on aphotographic representation of the specimen. The luminescencerepresentation indicates portions of the specimen where an activity ofinterest may be taking place. The photographic representation providesthe user with a pictorial reference of the specimen.

In-vivo imaging is performed by capturing an image of the sample using acamera. Intensified or cooled charge-coupled device (CCD) cameras areoften used to detect the localization of low intensity light-producingcells in the sample. These cameras are considerably complex, requirespecialized cooling, and are fixed to a single location on the top of aspecimen chamber. A user places a sample at a predetermined position onthe bottom of the specimen chamber within the field of view for theoverhead camera. This static relationship between camera and samplelimits image capture to overhead images only.

Often, it is desirable to capture different views of the sample. Forexample, the detection of internal light-producing cells from theunderside of a mammalian sample may be affected by covering tissue whichthe light must penetrate before being captured by the overhead camera.By gathering data from different angles, a user can obtain moreinformation about the location and intensity of a light source in theanimal than possible using only a single view. However, it may beimpractical to reposition the sample to capture a different view whenusing an overhead camera.

In view of the foregoing, an improved imaging system that enables thecapture of images from different views without repositioning the postureof the sample would be highly desirable.

SUMMARY OF THE INVENTION

The present invention relates to systems and methods for capturing animage of a sample. The sample is placed on a moveable stage in animaging box. The moveable stage allows an image of the sample, orportions thereof, to be captured by a camera from different views,angles, and positions within the imaging box without repositioning theposture of the sample. As the sample is variably located within theimaging box, a light transmission device assists image capture bytransmitting light emitted or reflected from the sample to a commondatum associated with a camera.

In one aspect, the present invention includes a processor which iselectrically coupled to the camera. The processor may also providecontrol of the moveable stage. In one embodiment, a transparent stage isused to allow image capture from angles beneath the stage.

In another configuration, the imaging system comprises an imaging boxhaving a set of walls enclosing an interior cavity. The imaging systemalso includes a camera mount configured to position the camera relativeto a fixed datum on one of the walls for viewing by the camera and alight transmission device. The imaging system additionally comprises amoveable stage apparatus including a transport mechanism and a stageconfigured to support the sample within the interior cavity. The stageis coupled to the transport mechanism for movement of the sample to oneof a plurality of positions in the interior cavity. The transportmechanism and the light transmission device cooperate to direct lightreflected or emitted from the sample to the fixed datum to capture theimage by the camera.

In another aspect, the imaging apparatus comprises an imaging boxincluding an interior cavity for receiving the sample and a stage forsupporting the sample. The imaging apparatus further comprises a firstlinear actuator attached to the imaging box and capable of positioningthe moveable stage in a first direction. The imaging apparatusadditionally comprises a second linear actuator attached to the firstlinear actuator, attached to the stage, and capable of positioning themoveable stage in a second direction. The first linear actuator and thesecond linear actuator cooperate to position the stage at one of aplurality of positions in the interior cavity.

In yet another aspect, the imaging apparatus includes a positioning armrotably coupled to the stage and rotably coupled to the imaging box suchthat the stage remains substantially horizontal for any rotationalposition of the positioning arm relative the imaging box. The imagingapparatus additionally includes a mirror attached to positioning arm.The mirror is configured to reflect light emitted from the sample atleast partially along a fixed datum.

In another aspect, the invention relates to a method for imaging asample. The sample is supported by a stage moveable within an imagingbox that is coupled to a camera configured to capture an image of thesample. The method includes moving the stage to a first position in theimaging box. The method also includes capturing a first image of thesample from the first position using the camera. The method furtherincludes moving the stage to a second position in the imaging box. Thesecond position has a different angle relative to a fixed datumassociated with the camera than the first position. The methodadditionally includes capturing a second image of the sample from thesecond position using the camera.

In still another aspect, the invention relates to a stage apparatus foruse with an imaging system for capturing an image of a sample with acamera. The imaging system includes an imaging box having a set of wallsdefining an interior cavity, and a camera mounted relative to a fixeddatum on one of the walls. The stage apparatus comprises a lighttransmission device, and a transport mechanism. The stage apparatusfurther includes a stage configured to support the sample within theinterior cavity where the stage is coupled to the transport mechanismfor movement of the sample to one of a plurality of positions in theinterior cavity. The transport mechanism and the light transmissiondevice cooperate to direct light reflected or emitted from the sample onthe stage to the fixed datum to capture the image by the camera.

These and other features of the present invention will be described inmore detail below in the detailed description of the invention and inconjunction with the following figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the figures of the accompanying drawings and in whichlike reference numerals refer to similar elements and in which:

FIG. 1A is a perspective view of an imaging system including an imagingbox adapted to capture images in accordance with one embodiment of theinvention.

FIG. 1B illustrates the structural components of the imaging box of FIG.1A in accordance with one embodiment of the present invention.

FIG. 2A illustrates a top perspective view of the components in the boxof FIG. 1A with the exterior walls removed showing the moveable stagedirectly below a fixed datum in accordance with one embodiment of thepresent invention.

FIG. 2B illustrates a top perspective view of the components in the boxof FIG. 1A with the exterior walls removed showing the moveable stagebelow and off-center from the fixed datum in accordance with oneembodiment of the present invention.

FIG. 2C illustrates a top perspective view of the components in the boxof FIG. 1A with the exterior walls removed showing the moveable stageabove and off center from the fixed datum in accordance with oneembodiment of the present invention.

FIG. 2D illustrates an internal side view of a side wall and housingincluded for the box of FIG. 1A in accordance with one embodiment of thepresent invention.

FIG. 2E illustrates an internal top perspective view of a side wall andhousing for the box of FIG. 1A in accordance with one embodiment of thepresent invention.

FIG. 2F illustrates a simplified view of light transmission within boxusing the light transmission device included in box of FIG. 1A.

FIGS. 3A and 3B illustrate a top and side view, respectively, of thestage included in the imaging box of FIG. 1A in accordance with oneembodiment of the present invention.

FIG. 3C illustrates a top perspective view of drawer and electroniccomponents housed therein in accordance with one embodiment of thepresent invention.

FIG. 4A illustrates a top perspective view of the components in the boxof FIG. 1A with the exterior walls removed showing the moveable stagedirectly below a fixed datum in accordance with another embodiment ofthe present invention.

FIG. 4B illustrates a top perspective view of the components in the boxof FIG. 1A with the exterior walls removed showing the moveable stagebelow and off-center from the fixed datum in accordance with anotherembodiment of the present invention.

FIG. 4C illustrates a top perspective view of the components in the boxof FIG. 1A with the exterior walls removed showing the moveable stageabove and off center from the fixed datum in accordance with anotherembodiment of the present invention.

FIG. 4D illustrates a gearing mechanism used to maintain the horizontalposition of the moveable stage of FIG. 4A in accordance with anotherembodiment of the present invention.

FIG. 5 is a process flow illustrating a method of capturing photographicand luminescence images using the imaging apparatus of FIG. 1A inaccordance with one embodiment of the present invention.

DETAILED DESCRIPTION

In the following detailed description of the present invention, numerousspecific embodiments are set forth in order to provide a thoroughunderstanding of the invention. However, as will be apparent to thoseskilled in the art, the present invention may be practiced without thesespecific details or by using alternate elements or processes. In otherinstances well known processes, procedures, components, and circuitshave not been described in detail so as not to unnecessarily obscureaspects of the present invention.

I. Imaging System

In one aspect, the present invention relates generally to improvedimaging systems. FIG. 1A illustrates an imaging system 10 adapted tocapture photographic and luminescence images in accordance with oneembodiment of the present invention. The system 10 provides userautomated control of image capture in an imaging box 12. The imagingsystem 10 is also useful for capturing and constructing structured lightimages.

The imaging system 10 comprises an imaging box 12 adapted to receive alight-emitting sample in which low intensity light, e.g.,luciferase-based luminescence, is to be detected. The imaging box 12includes a housing 16 on a side vertical wall of the box having a cameramount 109 (FIGS. 2A-2C) adapted to receive a camera. The imaging box 12is configured to be “light-tight”, i.e., essentially all external lightis prevented from entering the box 12 from the ambient room.

A high sensitivity camera, e.g., an intensified or a charge-coupleddevice (CCD) camera 20, is attached to the imaging box 12 preferablythrough the camera mount 109 affixed to the housing 16. The CCD camera20 is capable of capturing luminescent and photographic (i.e.,reflection based images) images of the sample within the imaging box 12.The CCD camera 20 may optionally be cooled by a suitable source such asa refrigeration device 22 that cycles a cryogenic fluid through the CCDcamera via conduits 24. A suitable refrigeration device is the“CRYOTIGER®” compressor, which can be obtained from IGC-APD CryogenicsInc., Allentown, Pa. Other refrigerants, such as liquid nitrogen orsolid state devices, may be used to cool the CCD camera 20.

An image processing unit 26 optionally interfaces between camera 20 anda computer 28 through cables 30 and 32, respectively. The computer 28,which may be of any suitable type, typically comprises a main unit 36that contains hardware including a processor, memory components such asrandom-access memory (RAM) and read-only memory (ROM), and disk drivecomponents (e.g., hard drive, CD, floppy drive, etc.). The computer 28also includes a display 38 and input devices such as a keyboard 40 andmouse 42. The computer 28 is in communication with various components inthe imaging box 12 via cable 34.

To provide communication and control for these components, the computer28 includes suitable processing hardware and software configured toprovide output for controlling any of the devices in the imaging box 12.The processing hardware and software may include an I/O card, controllogic for controlling any of the components of the imaging system 10,and a suitable graphical user interface for the imaging system 10. Thecomputer 28 also includes suitable processing hardware and software forthe camera 20 such as additional imaging hardware, software, and imageprocessing logic for processing information obtained by the camera 20.Components controlled by the computer 28 may include the camera 20, themotors responsible for camera 20 focus, one or more motors responsiblefor position control of a stage supporting the sample, the camera lens,f-stop, etc. The logic in computer 28 may take the form of software,hardware or a combination thereof. The computer 28 also communicateswith a display 38 for presenting imaging information to the user. By wayof example, the display 38 may be a monitor, which presents an imagemeasurement graphical user interface (GUI) that allows the user to viewimaging results and also acts an interface to control the imaging system10.

The processing hardware and software may also include a suitableprocessor configured to provide control signals to a motor coupled to amoveable stage included in box 12. The processor may also be configuredto prevent the stage from contacting the light transmission deviceduring movement of the stage. In addition to control functions, theprocessor may also be applied to perform various image processingfunctions described herein. For example, the processor may be configuredto produce a structured light representations using 2-D structured lightimages taken from one or more positions of the stage in the interiorcavity.

The imaging system 10 is suitable for capturing images from a variety ofviews and positions of the sample relative to the camera 20. Theseimages may be used in in-vivo imaging applications that include analysisof one or more representations of emissions from internal portions of aspecimen superimposed on a photographic representation of the specimen.In one embodiment, the imaging system 10 is used for 2-D and structuredlight imaging of a low intensity light source, such as luminescence fromluciferase-expressing cells, fluorescence from fluorescing molecules,and the like. The low intensity light source may be emitted from any ofa variety of light-emitting objects or samples which may include, forexample, tissue culture plates, multi-well plates (including 96, 384 and864 well plates), and animals or plants containing light-emittingmolecules, such as various mammalian subjects including mice containingluciferase expressing cells.

In one application, the sample is a biological specimen containing lightproducing cells. The resulting luminescence image may therefore becaptured without using any light sources other than the sample itself.Luminescence from the sample is recorded as a function of position toproduce the luminescence image. One approach to generating suchcomposite photographic/luminescence images is described in U.S. Pat. No.5,650,135 issued to Contag et al. on Jul. 22, 1997. The entiredisclosure of that patent is incorporated herein by reference for allpurposes.

In one particular embodiment, a 2-D luminescence image represents acollection of emitted photons received by each detector pixel of the CCDcamera 20 over a defined length of time. In other words, theluminescence image may display magnitude values representing the photoncounts at the individual detector pixels. Regions of the sample emittingradiation (e.g., photons) will appear in the luminescence image. Theluminescence images may indicate the presence of a biocompatible entity,for example. The entity can be a molecule, macromolecule, cell,microorganism, a particle or the like. Thus, an in-vivo analysis mayinclude detecting localization of a biocompatible entity in a mammaliansubject. Alternatively, the information in the live mode may be used totrack the localization of the entity over time. For more examples ofanalysis applications for a digital overlay image suitable for use withthe present invention, the reader is referred to in U.S. Pat. No.5,650,135, which was previously incorporated by reference.

II. Imaging Box

In one aspect, the present invention relates to an imaging apparatussuitable for various imaging operations. FIG. 1B illustrates theexternal components of imaging box 12 of FIG. 1A in accordance with oneembodiment of the present invention. FIGS. 2A-E and 4A-D illustrateinternal components of box 12 in accordance with various embodiments ofthe present invention. Each of the imaging apparatus described arecapable of capturing an image of a sample in box 12 using a cameracoupled thereto.

As shown in FIG. 1B, the imaging box 12 is illustrated with a door 18 inan open position, showing an interior cavity 44 for receiving thesample. The interior cavity 44 is defined by opposing side enclosurepanels 103 a and 103 b (103 b visible in FIG. 2D), a light-tightpartition 52 on the bottom, a top panel (not shown), a back enclosurepanel 47, and a front wall 48 defining a cavity opening 49 into theinterior cavity 44.

Below the cavity 44 is a smaller compartment separated therefrom by thelight-tight partition 52, the upper surface of which serves as a floorfor the cavity 44. In one embodiment, the smaller compartment provides ahousing space which is adapted to slideably receive a drawer 54 though afront opening 55 formed in the body 14. The drawer 54 houses electroniccomponents 56 which are in electrical communication with the computer 28(FIG. 1A) and control various components and functions of the box 14. Ina specific embodiment, the imaging box 12 has a body 14 made of asuitable metal such as steel.

A latchable door 18 is pivotally attached to box body 14 by way ofhinges 46 which permit the door 18 to be moved from the closed positionas shown in FIG. 1A to the open position as shown in FIG. 1B. In theopen position, door 18 enables user access to the cavity 44 through theopening 49. In the closed position, door 18 prevents access to thecavity interior 44 through the cavity opening 49.

Referring now primarily to FIGS. 2A-E, various internal components ofbox 12 (shown in broken lines) will now be described in accordance withone embodiment of the present invention. FIG. 2A is a top perspectiveview of the components in box 12 with the exterior walls removed showingstage 204 directly below fixed datum 107. FIG. 2B is a top perspectiveview of the components in box 12 with the exterior walls removed showingstage 204 below and off-center from fixed datum 107. FIG. 2C is a topperspective view of the components in box 12 with the exterior wallsremoved showing stage 204 above and off center from fixed datum 107.FIG. 2D is an internal side view of box 12 showing side wall 103 b andhousing 16 without light transmission device 111. FIG. 2E is an internaltop perspective view of side wall 103 b and housing 16 without lighttransmission device 111. FIGS. 2A-E are all shown with door 18 andexterior walls removed for illustration.

Referring to FIGS. 2C-2E, camera 20 is mounted to side housing 16 withthe camera lens 100 in view of interior cavity 44 through a port 101formed in side wall 103 b of box 12. The camera lens 100 is opticallycoupled to camera 20 of FIG. 1A and includes a user controlled apertureor F-stop ring 102 for adjusting the F-stop or aperture of lens 100,thereby modulating the amount of light passing through the lens. ANavitar, f 0.95, 50 mm TV lens is suitable for use as camera lens 100.The F-stop ring 102 includes circumferentially disposed teeth thatengage a gear 104 driven by an F-stop motor 105. The F-stop motor 105 isin electrical communication with the electrical components 56 andcontrolled by computer 28. Collectively, the motor 105 and a processorin computer 28 cooperate to position the f-stop of lens 100.

A focusing mechanism 160 (FIG. 2E) provides reciprocal movement of thelens for focusing thereof. The focusing mechanism includes a lenssupport 162 showing a stationary portion mounted to upper housing 16 anda movable portion that includes a threaded bore 113. A bolt 108,operably engageable with bore 113, includes a wheel that is driven by atoothed belt 110 through a corresponding drive wheel 112 of a cameralens focus motor 114 to move camera lens 100 into focus. The camera lensfocus motor 114 is in electrical communication with the electricalcomponents 56 and controlled by a processor included in computer 28 ofFIG. 1A.

A fixed datum represents a fixed region along the line of site of thecamera lens 100 into the interior cavity 44 of the box 12. Thus, thefixed datum 107 extends from the interior cavity in a directionsubstantially perpendicular to side wall 103 b and through the center ofcamera lens 100 (FIGS. 2A-E). This datum 107, for clarity, isrepresented by a stationary axis that provides a reference line of siteupon which the transport mechanism 202 and the light transmission device111 cooperate therebetween to direct light reflected or emitted fromsample 106 towards and into the camera lens 100 to capture images bycamera 20.

As shown in FIG. 2C, a camera mount 109 is attached to side housing 16of side wall 103 b. Camera mount 109 is adapted to receive and positioncamera 20 relative to fixed datum 107 for viewing of sample 106 withincavity 44 by camera 20. While camera 20 is capable of capturingphotographic images (i.e., reflection based images) of sample 106, it isalso sensitive enough to capture luminescence images thereof. Camera 20may employ a charge coupled device (CCD), a photodiode array, aphotogate array, or similar image capture device.

A moveable stage apparatus 200 is disposed in interior cavity 44, andincludes a transport mechanism 202 and a stage 204 to support thelight-emitting sample 106. Moveable stage apparatus 200 is capable oftwo degrees of freedom movement to reposition the stage 204 (and sample106) to a plurality of positions within interior cavity 44. Any oneposition therebetween may be retained for image capture.

As shown in FIGS. 2A-C, the transport mechanism 202 in the embodimentcomprises two linear actuators 206 and 208 oriented at substantiallyperpendicular to one another. Each linear actuator 206 and 208 iscapable of positioning stage 204 linearly along the respective actuator.Linear actuator 206 provides vertical positioning for stage 204 whilelinear actuator 208 provides horizontal positioning for stage 204.Linear actuator 206 has a stationary portion attached to box 12 and amobile portion attached to linear actuator 208. Linear actuator 208 hasa relatively stationary portion attached to linear actuator 206 and amobile portion attached to stage 204. An example of one such linearactuator suitable for use in the transport mechanism 202 is a LC-33produced by Thomson Industries of Port Washington, N.Y. Each linearactuator 206 and 208 also includes displacement limiting devices oneither end to restrict motion along their respective mobile portions.

The transport mechanism 202 preferably includes a set of positionsensors that are operably coupled to the computer 28 to provide positionfeedback to control the position of stage 204. In this case, theposition sensors include a string or thin string 144 having one endattached to the stage 204 while the other end is attached to a take-upreel 212 a (FIG. 2A). Based on the amount of string 144 wound on thereel and the total length of the string 144, computer 28 can determinethe length of string between the stage 204 and the sensor 142, (i.e.,based on changing resistance of the string with length and by using alook-up table in computer 28 to carry out the conversion). In anotherembodiment, the position sensor is provided by a laser positioned ininterior cavity 44 to intercept the moveable stage 204 at a startingvertical or horizontal position. The laser may then be used to calibratethe position of the moveable stage 58 to a common vertical or horizontalposition.

Linear actuators 206 and 208, position sensors 212, and computer 28combine to provide closed loop position control for stage 204 withininterior cavity 44. More specifically, a user, via computer 28, mayinput one or more positions for stage 204 along a substantially circularpath about fixed datum 107. In one embodiment, a user provides a viewingangle for stage 204 relative to fixed datum 107. Software included incomputer 28 then converts the viewing angle into control signals formoving each of the linear actuators 206 and 208. Motors included in eachof the two linear actuators 206 and 208 then receive the control signalsprovided by computer 28 and position stage 204 accordingly. The motionof stage 204 between image capture positions may be accomplished bysimultaneous motion of actuators 206 and 208 or by stepwise sequentialactivation of each of the actuators 206 and 208.

Light transmission device 111, as best reviewed in FIGS. 2A-2C, directslight reflected or emitted from sample 106 along the direction of fixeddatum 107 and into lens 100 for image capture by camera 20. Lighttransmission device 111 is mounted to housing 16 using stationarybracket 119 (FIG. 2A), which includes circumferentially disposedbearings between stationary bracket 119 and moving bracket 126 thatallow mirror assembly 120 to rotate freely relative to stationarybracket 119. Mirror assembly 120 is thus rotably coupled to housing 16and rotates about an axis co-axially aligned with the stationary axis ofthe fixed datum 107.

Referring to FIG. 2C, mirror assembly 120 comprises an angled mirror 121that reflects light from sample 106 on stage 204 in a direction alongfixed datum 107. Outer wall 123 is substantially cylindrical andincludes aperture 122 that enables light to pass between stage 204 andmirror 121. Outer wall 123 of mirror assembly 120 also prevents residuallight in interior cavity 44 not directly associated with the currentviewing angle of stage 204 from reaching lens 100. This is partiallyperformed by configuring mirror 121 to be sufficiently long to span thelength of stage 204. As the stage is positioned along the circular pathabout the stationary axis, outer wall 123 and mirror 121 cooperate tocollect light primarily from the angular direction of stage 204 which isthen reflected along fixed datum 107 for reception by lens 100.

FIG. 2F illustrates a simplified view of light transmission within box12 using light transmission device 111. As shown in FIG. 2F, for theposition of stage 204 as shown in FIG. 2A, light is emitted from sample106, reflected off mirror 121, and transmitted along fixed datum 107.

In one embodiment, a light source is provided within the barrel ofmirror assembly 120 to illuminate the sample or specimen in the imagingbox 12. The light source may be continuously illuminated or flashed tocapture photographic images of the sample and is turned off whencapturing luminescence images. In a specific embodiment, the lightsource comprises a ring of low-wattage lights disposed circumferentiallyaround the camera lens 100. In another embodiment, the light sourcecomprises four pairs of white-light emitting diodes (LEDs), one pairmounted in each of four corners around the camera lens 100. Oneadvantage of using LEDs is that the spectral emission thereof may becontained to visible light while excluding infrared light. Wires (notshown) may extend from the lights to the electronic components 56 andcomputer 28 to allow light levels to be controlled externally throughthe computer 28.

FIG. 2F also illustrates the use of structured light source 170. Asshown, structured light 175, emitted from structured light source 170,reflects off a mirror 173, passes through partially transparent mirror121, and onto sample 106. In one embodiment, the partial transparence ofmirror 121 is achieved using a half-silvered or partially silveredmirror. In another embodiment, a dichroic mirror having wavelengthspecific transparency properties is used. The structured light 175 maythen be captured by camera 20.

In the embodiment shown in FIGS. 2A-2E, light transmission device 111employs computer 28 to control and position mirror assembly 120 relativeto fixed datum 107. Mirror assembly 120 includes circumferentiallydisposed teeth on the inside of moving bracket 126 (teeth not shown)that engage a belt driven by a mirror assembly motor 128 (FIG. 2E).Moving bracket 126 then provides rotational motion relative tostationary bracket 119 for motor 128 input. Motor 128 is in electricalcommunication with the electrical components 56 and controlled bycomputer 28. Together, motor 128 and a processor in computer 28cooperated to control the rotary position of mirror assembly 120.

The two degrees of freedom movement provided by transport mechanism 202allow stage 204 and sample 106 to be positioned at multiple anglesrelative to fixed datum 107 for image capture by camera 20. Thus, basedon user input via computer 28, transport mechanism 202 and lighttransmission device 111 cooperate to direct light from sample 106 onstage 204 to fixed datum 107 and lens 100 to capture image using camera20. In addition to providing full 360 degree angular viewing of sample106 about the circular path, transport mechanism 202 is capable ofvarying the image depth for a given angle of stage 204 relative to fixeddatum 107. Together, transport mechanism 202 and light transmissiondevice 111 cooperate to provide a field of view for camera 20 in therange of about 7.5 cm to about 16.5 cm. In a specific embodiment, lighttransmission device 111 cooperate to provide a field of view for camera20 in the range of about 13 cm to about 16.5 cm. Similar to the userinitiated angular position control described above, a user may input adesired focal depth and viewing angle for stage 204. Software includedin computer 28 and linear actuators 206 and 208 would then combine toposition stage 204 at the desired angle and depth relative to fixeddatum 107.

To prevent undesirable contact between stage 204 and mirror assembly 120during operation, transport mechanism 202 may incorporate crashprotection measures. In one embodiment, the crash protection measuresare software based and controlled by a processor in computer 28. Thus,based on position feedback of stage 204 and known position of mirrorassembly 120, computer 28 generates control signals that insure thatstage 204 does not undesirably contact with mirror assembly 120. Thismay be advantageous for movement of stage 204 between a position such asthat shown in FIG. 2C and a position 180 degrees away. In this case, theprocessor of computer 28 transmits control signals to linear actuators206 and 208 which move stage 204 orbitally around mirror assembly 120,e.g., by maintaining a minimum radius from fixed datum 107.

Referring now to FIGS. 3A and 3B, a top and side view, respectively, ofstage 204 is illustrated in accordance with one embodiment of thepresent invention.

In one embodiment, stage 204 includes hardware based crash protectionmeasures that prevent undesirable contact between stage 204 and othercomponents within box 12. In a specific embodiment, crash pin 250 isplaced on the side of stage 204 closest to the camera 20, as shown inFIG. 3A. Crash pin 250 prevents contact between stage 204 and componentswithin cavity 44. To prevent contact between stage 204 and lighttransmission device 111, camera 20 or wall 103 b, a metal ring 260 isperimetrically disposed around light transmission device 111 onstationary bracket 119. Since metal crash pin 250 is ground and metalring 260 is maintained at 5V, inadvertent contact between crash pin 250and metal ring 260 acts as a limit switch and provides immediateelectrical communication with computer 28 that contact has been madewith stage 204. Movement of stage 204 is then stopped. Together, crashpin 250 and metal ring 260 provide a circular crash protection boundaryaround light transmission device 111 during movement of linear actuators206 and 208.

In another embodiment, software based crash protection may beimplemented for preventing undesirable stage 204 contact with componentswithin cavity 44. Based on position feedback of stage 204 using positionsensors 212 and known position of mirror assembly 120, computer 28provides control signals that ensure stage 204 does not overlap withmirror assembly 120, thus minimizing the risk of undesirable contactbetween sample 106 and components within cavity 44.

As shown in FIG. 3A, stage 204 comprises a frame 252 and a transparentportion 254. Transparent portion 254 allows light emitted or reflectedfrom sample 106 to be transmitted to light transmission device 111 withsubstantially no interference and minimal distortion for any position ofstage 204 about fixed datum 107. Transparent portion 254 preferably,comprises a transparent wire array 256 that supports sample 106. In aspecific embodiment, transparent wire array 256 is a single transparentnylon line interwoven through holes 258 on opposing edges of frame 252and secured in a taut manner to support sample 106. In anotherembodiment, array 256 is a mesh that resembles a cross pattern gridsimilar to a tennis racket mesh.

Box 12 may also include other components to facilitate image capture ofa sample within box 12. In addition to automated focus control of thecamera lens 100, the system 10 also includes an automated filter selectdevice 117 capable of selectively providing multiple filters 118 atleast partially between the camera 20 and light passing along fixeddatum 107. The filters 118 may each facilitate image capture for one ormore particular imaging applications. As shown in FIG. 2D, the opticalfilter select device 117 includes a circular filter select wheel 116adapted to carry a plurality of optical filters 118 around itsperimeter. The wheel 116 is rotatably mounted at its center to amounting bracket 130 attached to side housing 16. The filter wheel 116is mounted off-center from lens 100 such that the individual filters 118can each be rotated into position to intersect light emitted from thesample and reflected by mirror 121 before reaching the camera lens 100.Filter wheel 116 has a groove along its perimeter edge in which atoothed belt 131 is seated. The toothed belt 131 is also engaged with adrive wheel 134 on a filter wheel motor 136. The filter wheel motor 136is in electrical communication with the electrical components 56 andcontrolled by a processor included in computer 28. The plurality ofoptical filters 118 carried by filter wheel 116 may include any of avariety of optical filters for facilitating image capture such as aneutral density filter for bright samples, one or more wavelength cutofffilters for restricting specific wavelengths, a fluorescent filter forfluorescence applications in which the excitation light differs from thedetected light, etc.

Other components used to facilitate image capture of a sample within box12 may also include a gas manifold to anesthetize one or more mammaliansamples. In one embodiment, the gas manifold is detachably coupled tostage 204 and includes a plurality of interfaces. Each interface isadapted to provide a gas to a mammalian sample resting on the stage 204.An exemplary gas manifold suitable for use with the present invention isdescribed in commonly owned co-pending U.S. patent Ser. No. 09/795,056by Nelson et al. filed on Feb. 21, 2001, the entire disclosure of whichis incorporated herein by reference for all purposes.

Referring now to FIG. 3C, there is shown a top perspective view ofdrawer 54 and electronic components 56 housed therein. As previouslynoted, these components interface with computer 28 and are used tocontrol the various motors and other components of imaging system 10. A3 V power supply 137 provides electrical power to the various activecomponents in the drawer 54. A motor control board 146 has four motorcontrollers 148, 150, 152, 154 mounted thereon. The motor controllers148, 150, 152, 154 are in communication with each of the F-stop motor109, lens focus motor 114, filter wheel motor 136, mirror assembly motor128, and stage motor 138, respectively. Suitable control boards includethe TMG control board as provided by TMG of Mountain View, Calif. Eachmotor controller interfaces, via cable 34, with computer 28 where themotor controllers and motors are controlled by user input andappropriate software running on computer 28. Drawer 54 also houses adata acquisition board (DAB) 156. On the face of drawer 54 is a knob 155which is in communication with an interior cavity 44 light source andallows a user to manually to control the light intensity in the interiorcavity 44.

The F-stop motor 109, lens focus motor 114, mirror assembly motor 128,and filter wheel motor 136 are each stepper motors capable of suitableposition control of their respective components. By way of example, amodel number SST 39D 1010 (1.8 deg/step, 4.3V, 0.85 A), manufactured byShinano Kenshi Co., Ltd, Japan, is suitable for use with any of themotors 109, 114, 128 and 136. Each of the motors is in electricalcommunication with one or more electronic components 56 housed in drawer54. The electronic components 56 are, in turn, in communication with thecomputer 28 where the motors 109, 114, 128 and 136 may be controlled byappropriate software and/or by user input.

Referring now primarily to FIGS. 4A-C, an imaging apparatus forcapturing an image of sample 306 with camera 20 is illustrated inaccordance with another embodiment of the present invention. FIG. 4A isa top perspective view of the components in box 12 with the exteriorwalls removed showing stage 304 directly above fixed datum 307. FIG. 4Bis a top perspective view of the components in box 12 with the exteriorwalls removed showing stage 304 below and off-center from fixed datum307. FIG. 4C illustrates a top perspective view of the components in box12 with the exterior walls removed showing the moveable stage above andoff center from fixed datum 307.

Box 12 includes a camera lens 100 mounted on side housing 16 and coupledto camera 20, similar to that as described with respect to FIGS. 2C-2E.This datum 107, for clarity, is represented by a stationary axis thatprovides a reference line of site upon which the transport mechanism 202and the light transmission device 111 cooperate therebetween to directlight reflected or emitted from sample 106 towards and into the cameralens 100 to capture images by camera 20.

A moveable stage apparatus 300 is disposed in interior cavity 44, andincludes a transport mechanism 302 and a stage 304 to support thelight-emitting sample 306. Moveable stage apparatus 300 is capable oftwo degrees of freedom movement to reposition the stage 304 (and sample306) to a plurality of positions within interior cavity 44. Any oneposition therebetween may be retained for image capture.

As shown in FIGS. 4A-C, transport mechanism 302 rotates about main axis320 which passes through side wall 103 a. The center of rotation formain axis 320 which is co-axially aligned with the stationary axis offixed datum 307. Bearings are included between main axis 320 andsidewall 103 a which allow main axis 320 to rotate freely relative toside wall 103 a. A proximal end 320 a of main axis 320 is fixed to wormgear 325, which is operably driven by motor 324. A distal end 320 b ofmain axis 320 is fixed to a positioning arm 322 which supports themoveable stage apparatus 300. As motor 324 rotates worm gear 325, thisrotational motion is transmitted to the positioning arm 322 and themovable stage apparatus 300 for rotation about fixed datum 307. A S23Tas provided by Industrial Devices Corp. of Petaluma, Calif. is suitablefor use as motor 324.

Motor 324 is in electrical communication with electrical components 56and controlled by computer 28. Together, the motor 324 and the processorin computer 28 position movable stage apparatus 300 along the circularpath about fixed datum 307. An electrical slip ring 323 is provided toelectrically couple the components of box 12 to the stage mechanism tomaintain continuous electrical communication regardless of the rotationof positioning of main axis 320 without risk of wrapping. A AC4831-18 asprovided by Industrial Devices Corp. of Petaluma, Calif. is suitable foruse as electrical slip ring 323.

Positioning arm 322 provides the main structural support for movablestage apparatus 300 upon which stage 304 is rotably and slideablycoupled. Stage 304 is coupled to positioning arm 322 in a manner suchthat, as positioning arm 322 rotates via main axis 320 about fixed datum307, stage 304 remains substantially horizontal relative the bottom ofcavity 44. This allows a sample 306, which is supported atop stage 304,to be viewed from multiple positions and angles without falling offstage 304. To maintain the stage 304 in this horizontal position as thepositioning arm 322 rotates about main axis 320, a set of bevel gears350 a and 350 b are disposed between main axis 320 and a rod 330 thatrotably couples stage 304 to main support 322 (FIG. 4D). The bevel gears350 a and 350 b thus rotably couple main axis 320 to stage 304. Thebevel gears 350 a and 350 b reverse rotation received by rod 330 forrotation provided by main axis 320 in a 1:1 reverse gear ratio. Forexample, as main axis 320 rotates clockwise 30 degrees, rod 330 rotatescounterclockwise 30 degrees via bevel gears 350 a and 350 b, thuskeeping stage 304 horizontal. In this manner, stage 304 remainssubstantially horizontal for any rotation position of positioning arm322 relative to box 12, as shown in FIGS. 4B and 4C.

Centrally attached to main support 322 is light transmission device 311.Light transmission device 311 rotates with main support 322 about fixeddatum 307 and directs light reflected or emitted from sample 106 alongfixed datum 307 and towards lens 100 for image capture by camera 20.Light transmission device 311 includes two mirrors 335 and 336. Eachmirror 335 and 336 is attached to mirror support 339, which is fixed toand extends perpendicularly from positioning arm 322. Mirrors 335 and336 rotate with positioning arm 322 about fixed datum 307. Each mirror335 and 336 is configured to reflect light emitted or reflected fromsample 306 at least partially along fixed datum 307 and towards lens100.

Rotation about main axis 320 using motor 324 provides a first rotationaldegree freedom for movable stage apparatus 300. Movable stage apparatus300 also includes a second degree of freedom. More specifically, stage304 may translate linearly along positioning arm 322 towards and awayfrom mirrors 335 and 336 to vary the field of view for viewing of sample306 on stage 304. To allow linear translation of stage 304 alongpositioning arm 322, positioning arm 322 includes a linear slide 342which includes two cylindrical holes for receiving slide bars 342 a and342 b therethrough. Sliding mount 346 allows attachment by stage 304 tolinear slide 342. Sliding mount 346 is rotably coupled to linear slide342 via rod 330 and bearings disposed therebetween. Thus, stage 304 isorthogonally fixed to sliding mount 346, which rotates via rod 330 andtranslates via linear slide 342.

Motor 340 is capable of moving sliding mount 346 along slide bars 342 aand 342 b using a worm gear 349 operably coupled to motor 340 and linearslide 342. A SSD55D5C0D0 as provided by Shinano Kenski Co. of Japan issuitable for use as motor 340. Together, motor 340 and a processor incomputer 28 act to position stage 304 relative to mirrors 335 and 336 tocontrol the field of view for viewing of sample 306 on stage 304.

In operation, movable stage apparatus 300 and light transmission device311 may be used as follows. A user, via computer 28, inputs one or morepositions or angles for stage 304 relative to fixed datum 307. Forexample, the user may provide two viewing angles for stage 304 relativeto fixed datum 307, both having the same field of view. For the firstviewing angle, software included in computer 28 then converts theviewing angle into control signals for controlling motor 324. Motor 324then receives the control signals provided by computer 28 and positionsstage 304 at the first position having a first angle relative to fixedaxis 307. After imaging is complete from the first viewing angle,software included in computer 28 then sends control signals to motor324, which re-positions stage 304 at the second position having a secondangle relative to fixed axis 307.

Each mirror 335 and 336 is designed to provide a different field of viewfor imaging within cavity 44. Coupled with the ability to move stage 304towards and away from mirrors 335 and 336, mirror 335 provides a field aview in the range of about 15 cm to 25 cm. Similarly, mirror 336 a fielda view in the range of about 9 cm to 11 cm.

Similar to the stage embodiment in FIG. 3A, stage 304 comprises atransparent portion that allows light emitted or reflected from sample306 to be transmitted to light transmission device 311 withsubstantially no interference and minimal distortion for any position ofstage 304 about fixed datum 307. In addition, movable stage apparatus300 includes hardware based crash protection devices that preventundesirable contact between stage 304 and other components within box12. For example, slide 342 includes a hard stop at each end to preventmovement of stage 304 to undesirable positions along positioning arm322. Further, main axis 320 also includes a hard stop at the top centerthereof that prevents movable stage apparatus 300 from continuallycircling about main axis 320. Upon reaching the hard stop at top centerfrom a first direction, movement to the other side of the hard stop attop center may be accomplished by rotating the movable stage apparatusabout main axis 320 360 degrees in the opposite direction.

III. Operation of the Imaging System

The present invention may be employed in a wide variety of imagingapplications. Generally, the present invention may be applied with anynon-invasive methods and compositions for detecting, localizing andtracking light-emitting entities and biological events in a mammaliansubject. For example, the imaging system 10 may be implemented withintensified Charge-Coupled Device (CCD) cameras to detect thelocalization of light-producing cells (e.g., certain bacteria or tumorcells made bioluminescent by transforming them with luciferase DNAconstructs) inside of living animals, such as mice. In suchapplications, an animal containing the bioluminescent cells is placedinside of box 12 and on stage 204. Camera 20 is then activated to detectthe emitted photons. The photon signal may then be used to construct aluminescent image of photon emission. The luminescent image isconstructed without using light sources other than the luminescence fromthe sample itself. This luminescence is recorded as a function ofposition to produce the luminescence image. The photographic image mayalso be taken of the same sample to aid in position visualization of theluminescent image. One approach to generating such compositephotographic/luminescence images is described in U.S. Pat. No. 5,650,135issued to Contag et al. on Jul. 22, 1997. The entire disclosure of thatpatent was previously incorporated herein by reference.

Turning now to FIG. 5, process flow 500 illustrates a method ofcapturing photographic and luminescent images using the imaging system10 in accordance with one embodiment of the present invention. Processflow 500 begins by placing a specimen or assay to be imaged for lightemission on stage 204 within imaging box 12 (202). Using computer 28, auser inputs a desired position for stage 204. Based on the input,transport mechanism 202 moves stage 204 to the corresponding positionaccording to a control signal provided by computer 28 (504). Lighttransmission device 111 also re-positions according to a control signalprovided by computer 28. The imaging box 12 and associated imagecomponents are then prepared for photographic image capture of thesample (506). Preparation may include launching imaging and acquisitionsoftware (e.g., “LivingImage” as provided by Xenogen Corporation ofAlameda, Calif.) on the computer 28 and initializing camera 20. Furtherpreparations may include closing door 18, activating the photographiccapture option in the software, focusing camera 20 to a specific depthof the sample or animal, and turning on the lights in box 12.Preparations may also include focusing lens 100, selectively positioningan appropriate lens filter 118, setting the f-stop, etc.

A photographic image is then captured (508). In one embodiment, a “livemode” is used during photographic imaging of the sample to observe thesample in real time. The live mode includes a sequence of photographicimages taken frequently enough to simulate live video. Upon completionof photographic capture, the photographic image data is transferred toan image processing unit 26 and/or a processor in computer system 28(510). These may be used to manipulate and store the photographic imagedata as well as process the data for display on computer monitor 38.

Subsequently, with stage 204 at the same position, the imaging apparatus10 is prepared for luminescence image capture (512). Such preparationmay include selecting luminescent exposure time and binning level usingthe computer 28, and turning off the lights in interior cavity 44. Whenready, the CCD camera 20 then captures (514) the luminescence image overa set period of time (up to several minutes). The luminescence imagedata are transferred to the image processing unit 26 and/or a processorin computer 28 (516).

At this point, a user may manipulate and store the luminescence imagedata as well as process it for display on the computer display 38. Themanipulation may also include overlaying the luminescent image with thephotographic image and displaying the two images together as a 2-D“overlay” image, with the luminescence data typically shown inpseudocolor to show intensity. This overlay image may then be the basisfor user analysis and may be analyzed and manipulated as desired. Inparticular, an analysis may include a summation of the illuminationmagnitudes over the pixels within a portion of the luminescencerepresentation. Note that although the discussion will focus on a singleluminescence representation for the overlay image, the process flow 500may include taking multiple luminescence representations from the sameposition of stage 204, e.g., at the same time or a later time (518).

If desired, stage 204 may then be moved to a second position (520).While the stage is at the second position, one or more photographicand/or luminescence images of the sample may be captured as describedabove. Upon completion of each image capture, a processor in computer 28then receives the image data. Image collection may further continue bycapturing images of the sample from alternate positions and views of thesample.

As mentioned, the photon emission data may represent the specific pixelson the CCD camera 20 that detect photons over the duration of the imagecapture period. Together, a structured light photographic representationof the sample and a luminescence representation of the sample may becombined to form a structured light superposition or overlay image.Because the imaging apparatus 100 is typically used to measure theentire sample 106, the data in the luminescence representation typicallyhas one or more distinct luminescent portions of interest.

In one embodiment, the present invention includes the use of structuredlight during image capture. In this case, imaging apparatus 100 providesa sequence of images of a small animal containing a bioluminescentsource. This sequence of images is taken at different viewing angles andprovides the information necessary to reconstruct the location,brightness, and size of the bioluminescent source within the animal.Once the images are received by processor 28, one suitablereconstruction algorithm (or inversion algorithm) suitable for use withthe present invention is diffuse optical tomography. In order to applydiffuse optical tomography, it is necessary to determine the 3D surfacetopology of the animal and to map the bioluminescent emission onto thissurface. In one embodiment, 3D surface topology is accomplished using astructured light projection system.

Structured light uses a series of lines of light that are projected downon an object at an angle (at about 30 degrees, for example) to thesurface normal. The lines bend as they pass over the object, and thebend in the lines can be used to determine the height of the surface atall locations that are illuminated by a structured light projector 170.As shown in FIG. 2A, structured light projector 170 is attached to androtates with light transmission device 111. In this case, structuredlight projector 170 consists of a Kohler illumination system where aslide is illuminated by a light source and then an image of the slide isprojected onto the animal. The projection angle is large enough to getsufficient “bend” in the lines to achieve spatial resolution, but smallenough that large shadows are not present.

An image of the structured light is taken with camera 20. After the 2-Dstructured light images have been captured and stored, computer 28 maythen process the structured light data to generate a structured lightrepresentation (522). As one of skill in the art will appreciate, thereare numerous conventional algorithms for reconstructing a surface fromstructured light images. For example, the phase shift of each line atall points on the image can be determined from acomputationally-efficient 2D Fourier transform. The actual surfaceheight is then computed by “unwrapping” the phase map.

Each structured light image provides the surface topology forapproximately the facing half of the animal only. By taking images fromseveral viewing angles, e.g., about every 45 degrees, the entire 3Dsurface of the animal can be reconstructed by “stitching” together thepartial surface reconstructions obtained from each view.

Although the present invention has been discussed primarily in thecontext of a moveable stage useful for in-vivo imaging applications, thepresent invention is suitable for other imaging applications and may betailored correspondingly. In addition, although the present inventionhas been described with respect to an isolated box 12 and separatecomputer 28, one embodiment of the present invention relates to astand-alone cabinet unit housing all imaging components and computerprocessing components therein. Further, the present invention isscalable and may be adapted in size to fit to needs of a particularapplication. Although various details have been omitted for brevity'ssake, obvious design alternatives may be implemented. Therefore, thepresent examples are to be considered as illustrative and notrestrictive, and the invention is not to be limited to the details givenherein, but may be modified within the scope of the appended claims.

1. An imaging system for capturing an image of a sample with a camera,the imaging system comprising: an imaging box having a set of wallsenclosing an interior cavity; a camera mount configured to position thecamera within the interior cavity; a moveable stage apparatus includinga transport mechanism and a stage configured to support the samplewithin the interior cavity, the stage being coupled to the transportmechanism for movement of the sample to one of a plurality of positionsof the stage relative to the camera in the interior cavity, wherein thestage and the camera are configured to rotate relative to each other toa first position of the stage relative to the camera and to a secondposition of the stage relative to the camera; and a light transmissiondevice configured to direct light reflected or emitted from the sampleto the camera for each of the plurality of positions of the stagerelative to the camera in the interior cavity.
 2. The system of claim 1wherein the light transmission device comprises a mirror that reflectslight emitted from the sample towards a fixed datum.
 3. The system ofclaim 2 wherein the light transmission device is configured to rotateabout the camera.
 4. The system of claim 2 wherein the lighttransmission device is configured to rotate about the stage.
 5. Thesystem of claim 1 wherein the stage is configured to rotate about thecamera.
 6. The system of claim 5 wherein the stage is substantiallyhorizontal during rotation about the camera.
 7. The system of claim 5wherein the light transmission device is configured to rotate with thestage about the camera.
 8. The system of claim 1 wherein the cameramount is fixed on a wall of the set of walls.
 9. The system of claim 1wherein a portion of the stage that supports the sample is transparent.10. The system of claim 1 further including a structured light sourceconfigured to transmit structured light onto the sample.
 11. An imagingapparatus used in capturing an image of a sample, the imaging apparatuscomprising: an imaging box including an interior cavity for receivingthe sample; a stage for supporting the sample; a first linear actuatorattached to the imaging box and capable of positioning the moveablestage in a first direction; and a second linear actuator attached to thefirst linear actuator, attached to the stage, and capable of positioningthe moveable stage in a second direction, wherein the first linearactuator and the second linear actuator cooperate to position the stageat one of a plurality of positions in the interior cavity, and whereinthe stage and a camera are configured to rotate relative to each otherto a first position of the stage relative to the camera and to a secondposition of the stage relative to the camera.
 12. The apparatus of claim11 wherein the light transmission device comprises a mirror thatreflects light emitted from the sample towards the fixed datum.
 13. Theapparatus of claim 11 wherein the fixed datum is a fixed axisperpendicular to a vertical wall of the imaging box.
 14. The apparatusof claim 13 wherein the light reception device rotates about the fixedaxis.
 15. The apparatus of claim 11 wherein the first direction and thesecond direction are orthogonal.
 16. The apparatus of claim 15 whereinthe first linear actuator provides vertical positioning for the stageand the second linear actuator provides horizontal positioning for thestage.
 17. The apparatus of claim 11 wherein imaging box comprises acamera mount adapted to receive the camera, the camera capable ofcapturing an image of the sample within the interior cavity.
 18. Animaging system for capturing an image of a sample with a camera, theimaging system comprising: an imaging box having a set of wallsenclosing an interior cavity; a camera mount configured to position thecamera within the interior cavity; a moveable stage apparatus includinga transport mechanism and a stage configured to support the samplewithin the interior cavity, the stage being coupled to the transportmechanism for movement of the sample to one of a plurality of positionsin the interior cavity, wherein the plurality of positions include twodegrees of freedom relative to the camera; and a light transmissiondevice configured to direct light reflected or emitted from the sampleto the camera for each of the plurality of positions of the stagerelative to the camera in the interior cavity, wherein the stage and thecamera are configured to rotate relative to each other to a firstposition of the stage relative to the camera and to a second position ofthe stage relative to the camera.
 19. The system of claim 18 wherein thetransport mechanism and the light transmission device are configured tocooperate to direct light reflected or emitted from the sample to thecamera.
 20. The system of claim 19 wherein the light transmission devicecomprises a mirror that reflects light emitted from the sample towardsthe fixed datum.
 21. The system of claim 18 wherein the lighttransmission device is configured to rotate about the camera.
 22. Thesystem of claim 18 wherein the light transmission device is configuredto rotate about the stage.
 23. The system of claim 18 wherein the stageis configured to rotate about the camera.
 24. The system of claim 23wherein the stage is substantially horizontal during rotation about thecamera.
 25. The system of claim 18 wherein the camera mount is fixed ona wall of the set of walls.
 26. The system of claim 18 wherein a portionof the stage that supports the sample is transparent.